The major goal of this laboratory is to identify the molecular mechanisms responsible for producing salt-sensitive hypertension. The cloning of the epithelial Na channel (ENaC) has provided an outstanding opportunity to uncover the molecular mechanisms responsible for its regulation. Its importance in some forms of hereditary hypertension is well documented. By understanding how ENaC is regulated, molecular mechanisms responsible for more common forms of salt-sensitive hypertension can be directly addressed. This project will explore the mechanisms responsible for regulation of ENaC via the carboxy terminus of the alpha subunit. Published data from this laboratory and preliminary data presented in this application suggest that specific regions of this carboxy terminus can be involved in either an increased or a decreased activity of the channel. The proposed experiments seek to determine proteins that interact with the carboxy terminus of the alpha subunit that will increase its activity. Using candidate proteins they have identified by a yeast-2-hybrid screen, the investigators will characterize their interactions with the alpha-ENaC carboxy terminus. Several assays will be used to determine the specific interactions and mutations will be generated to determine the critical regions for these interaction. The effects of identified proteins on ENaC function will be tested in parallel using heterologous expression systems, including Xenopus oocytes and epithelial cells expressing endogenous ENaC. Interactions with endogenous kinases will be examined using inhibitors and specific mutations. The aim of the project is to identify those proteins that can interact with specific regions of the carboxy terminus of the alpha subunit and to construct a model of kinase mediated increase in ENaC function.